Asexual propagation of plants has been shown for some species to yield large numbers of genetically identical embryos, each having a capacity to develop into a normal plant. Such embryos are usually further cultured under laboratory conditions until they reach an autotrophic “seedling” state characterized by an ability to produce their own food via photosynthesis, resist desiccation, produce roots able to penetrate soil, and fend off soil microorganisms. Some researchers have experimented with the production of artificial seeds, known as manufactured seeds, in which individual plant somatic or zygotic embryos are encapsulated in a seed coat. Examples of such manufactured seeds are disclosed in U.S. Pat. No. 5,701,699, issued to Carlson et al., the disclosure of which is hereby expressly incorporated by reference.
An embryo suspension mass (“ESM”) is used to cultivate embryos that are subsequently inserted into manufactured seeds. Current somatic embryogenesis involves the ongoing maintenance and planting of ESM having diverse mixtures of cell clump sizes and types. The inventors of the current disclosure have discovered that small, usually single cells from the ESM generally have a higher yield than larger cells. That is, small cells tend to have a higher rate of survival and ultimately germinate into healthy plants or trees.
Currently available methods for size separation include passively pouring cell culture onto a sieve or set of nested sieves. Another currently available method is density-based centrifugation where cell culture is disposed in a test tube with a separation media, such as Percoll or Ficoll. The test tubes are centrifuged to segregate the cells on the basis of density.
Although such methods for size separation are effective, they are not without their problems. As a non-limiting example, such methods often subject the cells to contamination. Additionally, when centrifugation is included as part of the density separation, the centrifuge often causes shearing and compaction damage to the cells.
Thus, there exists a need for a method of separating an ESM that is capable of reliably separating the ESM at a relatively low cost, and minimizing the risk of damaging or contaminating the ESM.